Thomas Søndergaard Stenum:
Roles of tRNA and tRNA-related RNAs in the regulation of protein synthesis in Escherichia coli

Date: 12-11-2018    Supervisor: Michael Askvad Sørensen & Sine Lo Svenningsen

The present PhD thesis provides new insights into the regulation and importance of tRNA levels in Escherichia coli. Moreover, it describes the identification of a novel group of small RNAs transcribed as part of the ribosomal RNA primary transcript.

We find that the majority of the tRNA pool is degraded twenty minutes after the onset of single amino acid starvation. This decreased stability of tRNA is independent of the stringent response as a mutant strain not able to make (p)ppGpp also degrade tRNA as a consequence of amino acid starvation. Furthermore, we show that this degradation does not require de novo synthesis of effectors to take place. Based on these results we present a demand-based model, in which tRNAs are subjects for degradation whenever they are not occupied by the translational machinery. The method used to purify and quantify tRNAs is described in detail and accompanied by a video showing how to perform each individual step. For precise and unbiased quantification of tRNA levels this method relies on the addition of spike-in cells. Furthermore, the thesis presents evidence of a novel group of sRNA located downstream of three out of seven ribosomal RNA operons in E. coli. These sRNAs belong to the larger group of sequences the "tRNA-linked repeats" (TLRs). We show that these three TLRs bind to both the RNA chaperone Hfq and the post-transcriptional regulator CsrA. We present evidence that Hfq assists in folding the TLRs and possibly also guides their processing. Altered expression of these TLRs affect various CsrA-related phenotypes and we argue that they constitute a novel group of CsrA regulators. Lastly, we investigated an E. coli mutant lacking three out of four gene copies encoding the major arginine accepting tRNA. We find that this deletion leads to an approximately 4-fold increase in fimA expression, the gene encoding the major type 1 fimbriae subunit. Besides this, 36 genes were found to be more than 2-fold differentially expressed in the mutant compared to the wild type. Of these, all but four were also found to be differentially expressed in a csrA mutant. We propose a model in which the arginine accepting tRNAs regulate the expression of fimA, which in turn regulates the activity of CsrA by binding and titration. The mechanics behind fimA regulation by arginine accepting tRNA is unclear, however, based on phenotypic complementation by expression of a mutated tRNA, we suggest that the sequence of the tRNA is important rather than its' direct role in translation.